The anthracyclines of the daunorubicin group, such as doxorubicin, carminomycin and aclacinomycin, are among the most widely employed agents in antitumoral therapy [F. Arcamone, Doxorubicin, Academic Press, New York, 1981, pp. 12-25; A. Grein, Process Biochem. 16:34 (1981); T. Kaneko, Chimicaoggi May 11, (1988)]. Improved derivatives of daunorubicin and doxorubicin have been made by chemical synthesis to enhance their antitumor activity, particularly by the oral route of administration, and to combat the acute toxicity and chronic cardiotoxicity associated with the use of these drugs in the treatment of cancer [Penco, Process Biochem. 15:12 (1980); T. Kaneko, Chimicaoggi May 11, (1988)]. 4'-Epidoxorubicin (Epirubicin.RTM.) and 4-demethoxydaunorubicin (Idarubicin.RTM.) are examples of such analogs.
These naturally occuring compounds are produced by various strains of Streptomyces (S. peucetius, S. coeruleorubidus, S. galilaeus, S. griseus, S. griseoruber, S. insignis, S. viridochromogenes, S. bifurcus and Streptomyces sp. strain C5) and by Actinomyces carminata. Doxorubicin is only produced by S. peucetius subsp. caesius but daunorubicin is produced by S. peucetius as well as the other Streptomyces described above. The type strains S. peucetius subsp. caesius IMRU 3920 (this strain is the same as ATCC 27952 and hereinafter is abbreviated to "S. peucetius 3920"), S. peucetius ATCC 29050 ("S. peucetius 29050"), and S. peucetius subsp. caesius ATCC 27952 ("S. peucetius 27952") are publically available and are described in U.S. Pat. No. 3,590,028. S. peucstius 29050 and 27952 have been deposited at the American Type Culture Collection, Rockville, Md. USA, receiving the index number ATCC 29050 and 27952.
The anthracycline doxorubicin (2) is made by S. peucetius 27952 from malonic acid, propionic acid, and glucose by the pathway shown in FIG. 1 of the accompanying drawings. .epsilon.-Rhodomycinone (4), carminomycin (3) and daunorubicin (1) are established intermediates in this process [Grein, Advan. Appl. Microbiol. 32:203 (1987), Eckardt and Wagner, J. Basic Microbiol. 28:137 (1988)]. Two steps in this pathway involve the O-methylation of discrete intermediates: the conversion of aklanonic acid to methyl aklanonate and carminomycin (3) to daunorubicin (1). Cell-free extracts of S. peucetius 29050, S. insignis ATCC 31913, S. coeruleorubidus ATCC 31276 and Streptomyces sp. C5 have been shown to catalyze the latter step in the presence of S-adenosyl-L-methionine [Connors et al., J. Gen. Microbiol. 136:1895 (1990)], suggesting that all of these strains contain a specific carminomycin 4-O-methyltransferase (COMT protein).
Genes for daunorubicin biosynthesis and daunorubicin resistance have been obtained from S. peucetius 29050 and S. peucetius 27952 by cloning experiments [Stutzman-Engwall and Hutchinson, Proc. Natl. Acad. Sci. USA 86:3135 (1988); Otten et al., J. Bacteriol. 172:3427 (1990)]. These studies have shown that, when introduced into Streptomyces lividans 1326, these cloned genes confer the ability to produce .epsilon.-rhodomycinone and to become resistant to daunorubicin and doxorubicin to this host. In subsequent work we examined whether these clones could confer the ability to convert carminomycin to daunorubicin when introduced into S. lividans. We have now isolated a 1.6 kilobase (kb) DNA segment that incorporates the carminomycin 4-O-methyltransferase gene, which hereinafter will be abbreviated as "dnrK".